1. Field of the Invention
The present invention relates to equipment and methods for assaying the amount of optical fluorescence, and the degree of fluorescence polarization, in samples.
2. Background and Description of the Related Art
Fluorescence Methods and Terminology.
Fluorescence involves exciting a molecular group with light of a first wavelength, causing it subsequently to emit light of a second, longer wavelength. The molecular group is termed a fluorophore, and the first and second types of light are termed xe2x80x9cexcitationxe2x80x9d and xe2x80x9cemissionxe2x80x9d light, respectively. Between excitation and emission, the molecular group is said to be in an excited state. Depending on the molecular group involved, the time spent in the excited state can vary widely, from a few nanoseconds to several microseconds. The duration of the excited state is termed the fluorescence lifetime. It is common to chemically engineer a fluorescent marker compound by grafting a fluorophore to a chemical group that reacts only or primarily with a very specific target molecule. The resultant fluorescent marker will bind only to very specific targets, and has fluorescence properties of the fluorophore.
Fluorescent markers are used to disclose the presence and/or location of targets within a sample, which may contain a variety of other compounds. For reliable detection, the other compounds in the sample must exhibit a very low degree of fluorescence, or there must be a way to discriminate between fluorescence emission resulting from the target compound and that from other compounds in the sample. Since the mechanism of fluorescence is present to at least some degree in most compounds, discrimination means are usually employed. Among the means are discrimination by wavelength of excitation light, by wavelength of emission light, and by fluorescence lifetime. Typically, discrimination by excitation wavelength involves making measurements using excitation light that has been filtered to contain light of only a selected wavelength band. Similarly, measurement of emission light through a filter that admits only a selected wavelength band provides a means to discriminate by emission wavelength.
Fluorescence lifetime discrimination is performed by a variety of methods, the simplest of which is to excite using a modulated source, and to observe emission using synchronous detection methods. For example, a target compound with a long fluorescence lifetime may be detected by exciting the sample with brief pulses, while measuring emission using a gated detector which is insensitive for a controlled, brief interval after excitation. Such a detector will not respond to compounds having a short fluorescence lifetime, which will have ceased emission by the time the detector becomes responsive. However, the target species, having a long fluorescence lifetime, will continue to emit for considerably longer, and the majority of its emission will be detected. Alternative approaches can also be used with single-element detectors, including detection with lock-in amplifiers, quadrature detection, and other standard signal analysis techniques. Imaging detection is possible with a gated intensifier or microchannel plate (MCP), by electronic shuttering, or by pixel shifting between photosensitive and non-photosensitive regions of a CCD detector.
Discrimination by these means is useful for removing so-called xe2x80x98backgroundxe2x80x99 fluorescence arising from optical components, solvents, culture dishes, and the like, which are necessary elements in a fluorescence experiment but whose fluorescence signal is not seen as contributing meaningful information. That is, these are means for removing unwanted contaminant signals and for obtaining an enhanced signal-to-noise ratio in the sample fluorescence measurement.
It is possible to use discrimination techniques not just to remove background, but to learn more about the sample itself. For example, two or more fluorescent markers can be used which have distinct excitation or emission properties, so a single sample can be tagged with multiple markers to identify different structures or entities. The fluorescent signals are then resolved to obtain information about each marker independently. This technique is often termed multiprobe fluorescence.
Discrimination by excitation or emission wavelength may also be used to learn additional information about a single target species. For some fluorescent markers, the characteristic wavelengths of optimal excitation or emission vary with the chemical properties of the environment, such as the pH, salinity, concentration of calcium, or the presence of other, very specific molecules. Observing how fluorescence intensity varies with excitation wavelength, or measuring the spectrum of emission light, can provide a measurement of the chemical environment of the fluorescent marker. These practices are termed fluorescence excitation spectroscopy and fluorescence emission spectroscopy.
One case of special significance is fluorescence resonance energy transfer (FRET), which employs two molecular groups having carefully related properties. Typically, one group contains a fluorophore that is characteristically excited at a first wavelength and emits at a second wavelength. The other group is characteristically excited at the second wavelength and emits at a third wavelength. Depending on the presence, concentration, or molecular conformation of the two groups, the likelihood of interaction between the two groups is higher or lower. When the likelihood of interaction is high, energy is transferred from excited molecules in the first molecular group to the second molecular group, resulting in emission light at the third wavelength. Thus, sample emission at the third wavelength is enhanced and emission at the second wavelength is depressed, compared to the case when the likelihood of interaction is low. Typically, a FRET experiment involves excitation at the first wavelength while monitoring the level of emission at the second and third wavelength bands. From the ratio of emission at these bands, the level of interaction is inferred.
Additional information can be obtained in some cases by analyzing the fluorescence polarization (FP), which involves exciting the sample with linearly polarized light and measuring the degree of linear polarization in the emission light. Excitation light preferentially excites those molecules having a selected geometrical orientation relative to the light""s polarization vector. Thus, the population of excited molecules is selectively oriented, rather than randomly so, at the time of excitation. If the fluorescence lifetime is comparable to or shorter than the molecular reorientation time, then the molecules will also be preferentially oriented when they emit, and the emission light will be linearly polarized to some degree. By measuring the degree of polarization, one infers the degree of preferential orientation at time of emission. It is conventional to refer to the degree of polarization (DOP) in terms of millipolarization units (MPU), defined as
DOP=1000*(I∥xe2x88x92I195)/(I∥+Ixe2x8axa5)xe2x80x83xe2x80x83[1]
where I∥ and Ixe2x8axa5 are the intensities of fluorescence emission polarized in the same sense as the excitation light and polarized orthogonal to it, respectively. Related measures are also used to quantify fluorescence polarization, based on substantially the same information.
Many factors can affect molecular re-orientation time, including rotational viscosity, temperature, and whether the fluorescent molecule is bound to another molecule or not. Measurement of fluorescence polarization (FP) is often used as a way to assess whether a molecule is in its bound or free state.
It is common to use mercury or xenon arc lamps with optical filters for fluorescence excitation. These are very useful for providing ultraviolet (UV) light, which some fluorophores require. When only visible light is required for excitation, a less expensive tungsten lamp can be used instead. Most often, lamp sources are used when fine definition of the illumination region is not required, or when a large region is to be illuminated. Laser sources are sometimes employed to illuminate small, well-defined regions, because of their higher specific radiance and more readily controlled beam properties. For example, lasers are often used as excitation sources in confocal equipment, and to create very high flux densities in e.g. single-molecule detection experiments. They are limited in that they emit a restricted, often discrete set of wavelengths in contrast to lamps, which generally produce a continuous spectrum that can be filtered to provide any desired band within a range.
Detection of fluorescence in multiple-sample assay plates is typically performed by optical scanning means, by sequential measurements with single-element instruments that are mechanically stepped across the individual sample regions, or by use of array detectors with flood-light sources,. Examples of optical scanning means include Kain, in U.S. Pat. Nos. 5,672,880 and 5,719, 391, where a galvo-driven scanning mirror directs excitation light toward, and emission light from, one of a plurality of samples. This allows a single lamp and a single detector to be used, and minimizes the number of moving parts required.
In systems such as the Analyst from LJL Biosystems (Sunnyvale, Calif.), a single-element reader is mechanically stepped across the various samples in a sample plate. The Analyst arrangement enables use of a specialized optical design where the excitation and emission optics define a sample volume in an approximately confocal arrangement, with different optical axes angles. However, readout time and instrument cost are increased because of the need for mechanical stepping.
These approaches use a single excitation source and a single detector when measuring total fluorescence levels. Typical detector types include avalanche photodiodes (APD), photomultiplier tubes (PMT), or other devices capable of operating at low light levels. This is important, as high sensitivity is vital to fluorescence assay instruments. And since only a single detector is required, the cost or complexity of that element can be increased somewhat without great consequence.
In contrast, other systems use an array detector such as a silicon charge-coupled device (CCD) or photodiode array to measure fluorescence from many samples at once. This parallelism increases the instrument throughput, and because modem CCD detectors have low-noise readout circuitry, the detector does not impose a significant penalty in terms of reduced sensitivity. The Arthur system from E.G.andG. Wallac (Gaithersburg, Md.) utilizes a flood source to illuminate many samples at once, and a CCD sensor to measure the fluorescence emission.
For FP analysis, additional components are employed. It is necessary to polarize the excitation source, which is readily achieved with conventional polarization optical elements for the visible and UV, such as dichroic sheet polarizer, polarizing beamsplitter cubes, and crystal polarizers such as Glan-Taylor or Rochon prisms.
The polarization state of the emission light is analyzed using one of several approaches. In one class of FP instruments, emission light is analyzed using a linear polarizer that is rotated to two orthogonal settings while a detector is read, to measure the components I∥ and Ixe2x8axa5. Typically a sheet dichroic type of polarizer is used, but use of any linear polarizer would result in a similar overall function. This type of instrument, referred to in the present application as a sequential-measurement FP reader, has a number of drawbacks. Because it measures the two states in time-sequence rather than simultaneously, its accuracy is degraded by fluctuations in the lamp or laser source. One can employ a reference detector to monitor the source fluctuations, and numerically compensate for variations by e.g. division, but this approach is not entirely successful. Further, intrinsic changes in the sample during the process of measurement, such as the effects of photobleaching, cannot be corrected. Finally, mechanical moving parts are normally used to select alternating polarization states, introducing reliability concerns.
Another class of FP instruments uses a polarizing beamsplitter (PBS). This separates the fluorescent emissions into two distinct beams according to their polarization state, and these beams are directed onto separate detectors to measure the components I∥ and Ixe2x8axa5. This type of instrument is termed a simultaneous-measurement FP reader in the present application. It measures both states simultaneously, and so does not suffer the problems of the sequential-measurement FP reader just described. However, it has other limitations. A PBS is a pair of right-triangle prisms with optical coatings on the hypotenuse, at which face they are cemented or joined to form a cube. This means that the two detectors must be physically distinct parts, rather than being two segments of a multi-element detector, because the images formed by the two beams are not coplanar. The need for two detectors, two sets of readout circuitry, and sometimes two lenses, means increased cost and complexity.
Both of the FP instrument designs described above read a single sample at a time, and no known commercial FP instrument can read a plurality of samples at once. This is a weakness for applications such as clinical testing and high-throughput drug screening, since single-sample systems inevitably have lower sample throughput. While one can conceive of an instrument based upon an array of detectors, polarizers, and sources, the construction of an instrument with competitive price and performance to existing single-sample instruments has not been achieved. This is a significant limitation of the prior-art designs. A system described in U.S. Pat. No. 5,943,129 to Hoyt and Miller could be used for detection of multiple samples within an assay plate, and can select alternating polarization states using liquid crystal elements means rather than mechanical means. However, it is a sequential-measurement FP reader with all the inherent problems of this approach, as explained above.
Another inherent limitation of all prior-art systems is their need for calibration. There is generally some polarization dependence in the transmission of lenses, in the reflection from mirrors, and the like. So, the amount of light reaching the detector(s) is altered by these elements, which are normally present in a practical system. For a simultaneous-reading PBS-type instrument, this is compounded by the fact that the two detectors employed generally have somewhat different quantum efficiencies, and are measured by different electronic circuits. Due to the differences in the optical elements when transmitting the two types of polarized light, and possible detector differences, the system has different responsivity for measurement of I∥ and of Ixe2x8axa5. Since there is no way to assess the relative proportions of I∥ and Ixe2x8axa5, this voids the measurement of FP unless the system can be calibrated.
The factors producing disparate responsivity between I∥ and Ixe2x8axa5 are not necessarily constant in time, nor are they the same at all wavelengths. Consequently, calibration must be undertaken separately for each wavelength band of fluorescent emission, and must be repeated at intervals to accommodate aging in components and circuitry. These problems are most severe when two detectors are used, as in a simultaneous measuring system.
In summary, the aforementioned art includes fluorescence measurement instrumentation for optically scanning a plurality of samples on a plate, or mechanically stepping a single-point system to a plurality of samples, or for flood-illuminating a plate and imaging a plurality of samples using a CCD detector. It provides methods for measuring the degree of fluorescence polarization through simultaneous-measurement or time-sequential measurement of orthogonally polarized emission components, but all suffer from significant limitations. All require calibration to obtain high-accuracy readings. Those which employ simultaneous-measurement of orthogonally polarized fluxes have higher cost and parts count, while those employing sequential-measurements often require moving parts; they are sensitive to fluctuations in the lamp or laser used to excite fluorescence; and their accuracy can be compromised by the inevitable photobleaching of the sample itself. Nor does any prior art system enable measuring the fluorescence polarization of many samples at once. Thus, no prior art system provides a self-calibrated measurement of fluorescence polarization, with high accuracy and the capability to measure many samples at once, for reading multiwell plates, microscope slide samples, and similar applications.
It is an object of the present invention to describe a means for measuring fluorescence which can read many samples at once, in parallel, for high throughput screening. A further object is to enable reading plates, pipettes, tubes, microscope slides, and a wide variety of formats with little or no change to the instrument hardware. Another object is to provide for measurement of multi-band fluorescence, time-resolved fluorescence, fluorescence emission spectroscopy, and fluorescence excitation spectroscopy in a single instrument. A further object is to provide enhanced sensitivity measurements, to enable use of smaller sample sizes and lower concentrations. Yet another object is to provide a method and means for fluorescence polarization measurements which are inherently accurate with no need for calibration, and which do not suffer degraded accuracy despite fluctuations in the excitation source. A final object is to provide means and methods for improving the accuracy and sensitivity of fluorescence polarization measurements made with existing fluorescence instrumentation.
Other objects and features of the present invention will become apparent from the following detailed description considered in conjunction with the accompanying drawings. It is to be understood, however, that the drawings are designed solely for purposes of illustration and not as a definition of the limits of the invention, for which reference should be made to the appended claims.
The various features of novelty which characterize the invention are pointed out with particularity in the claims annexed to and forming a part of the disclosure. For a better understanding of the invention, its operating advantages, and specific objects attained by its use, reference should be had to the drawing and descriptive matter in which there are illustrated and described preferred embodiments of the invention.
The present invention provides for a fluorescence measurement instrument comprising excitation means, a plurality of sample regions, and detection means; where the excitation means produce a first beam and a diffractive optical beamsplitter element that splits the first beam into plural secondary beams; the plural secondary beams excite the plurality of sample regions simultaneously to effect fluorescence; and the detection means detect the fluorescence from the plurality of sample regions.
The present invention further provides for a fluorescence measurement instrument comprising excitation means, a sample region, and detection means; where the excitation means and the detection means comprise an objective and a photodetector; where light from the laser source is directed toward the sample region by a mirror located between the sample region and the objective.
The present invention additionally provides for a fluorescence polarization measurement instrument comprising excitation means, at least one sample region, and detection means; where the excitation means produce light that is substantially linearly polarized along a first axis of polarization at the sample region; where the detection means comprise an objective, a photodetector, and a polarization analyzer; where the photodetector provides a plurality of spatially distinct pixel regions; where the objective directs a beam of fluorescent light from the sample toward the polarizing beamsplitter; where the polarization analyzer divides the beam of fluorescent light into two linearly polarized secondary beams, one with polarization axis oriented substantially parallel to the first axis of polarization and the other with polarization axis oriented substantially perpendicular to the first axis of polarization; and where the secondary beams of fluorescent light are directed onto the spatially distinct pixel regions of the photodetector by the polarization analyzer.
Another embodiment of the present invention is a fluorescence polarization measurement instrument comprising excitation means, at least one sample region, and a detection means; where the excitation means produce light that is directed at the sample region to effect fluorescent emission and that is substantially linearly polarized along a first axis of polarization at the sample region; where the detection means comprise an objective, a plurality of independent detector regions, and a polarization analyzer; where the plurality of independent detector regions comprises one of a unitary detector with multiple pixel regions and multiple detectors; where the objective collects the fluorescent emission from the sample region and directs the fluorescent emission in a beam toward the polarization analyzer; where the polarization analyzer divides the beam of fluorescent emission into two linearly polarized secondary beams, one with polarization axis oriented substantially parallel to the first axis of polarization and the other with polarization axis oriented substantially perpendicular to the first axis of polarization; where the linearly polarized secondary beams are directed by the analyzer to separate detector regions; where said excitation means further provide switching means for changing the state of polarization of the excitation light at the sample region during a single fluorescence polarization measurement from a first orientation parallel to the first axis of polarization to a second orientation parallel to a second axis of polarization which is substantially perpendicular to the first axis of polarization.
Finally, the present invention provides for a method of measuring fluorescence polarization, consisting of illuminating a sample to effect fluorescence emission with a beam of excitation light that is linearly polarized along a first axis measuring the intensities of a first component of the fluorescence emission that is polarized along the first axis and a second component of the fluorescence emission that is polarized orthogonal to the first axis while the sample is illuminated with the beam of excitation light that is linearly polarized along the first axis; switching the state of polarization of the beam of excitation light to be linearly polarized along a second axis substantially orthogonal to the first axis; measuring the intensities of a third component of fluorescence emission that is polarized along the first axis and a fourth component that is polarized orthogonal to the first axis while the sample is illuminated with the beam that is linearly polarized along the second axis; calculating the fluorescence polarization based on the measurements of the intensities of the first, second, third and fourth components.